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Delayed flow-through cytoplasm of newly synthesized Balbiani ring 75S RNA
Cell, Vol. 13, 727-733,
April
1978,
Copyright
0 1978 by MIT
Delayed Flow-Through Cytoplasm of Newly Synthesized Balbiani Ring 7% RNA U. Lonn Department of Histology Karolinska Institute S-104 01 Stockholm 60, Sweden
Summary With a nonaqueous microdissection technique, the cytoplasm of Chironomus salivary gland cells can be separated into concentric zones situated at increasing distances from the nuclear envelope. This dissection technique is used here to investigate the cytoplasmic distribution of 75s RNA of Balbiani ring origin. The Balbiani ring 7% RNA has properties of a messenger RNA coding for secretory proteins. After a pulse of RNA precursor to the living animal, labeled Balbiani ring 75s RNA is found mainly in the cytoplasm located closer to the nuclear envelope, with smaller amounts toward the periphery of the cell. This gradient, initially very steep, lasts for at least 2 days, but less than 6 days. Experiments with 5fluorouridine indicate that the formation of the gradient does not depend upon simultaneous export of ribosomal subunits. After a pretreatment of the animals with the protein synthesis inhibitor cycloheximide, however, newly synthesized 7% RNA distributes evenly in the cytoplasm-that is, this treatment prevents the formation of the 75s RNA gradient. The gradient in salivary glands of normally cultured animals is therefore likely to be the result of diffusion restriction of the labeled 75s RNA. Thus the 75s RNA located closer to the nuclear envelope is the most recently exported 755 RNA. An explanation of these results is that the 75s RNA associates with the membranes of the endoplasmic reticulum early or immediately after nuclear release. This association should occur in the cytoplasm surrounding the nucleus and may occur either as single particles and/or as parts of polysomes. Introduction Proteins destined for export from the cell, such as secretory proteins, are synthesized on polysomes associated with the membranes of the endoplasmic reticulum (ER). Such polysomes also synthesize proteins that are destined for inclusion in other membrane-bound organelles as well as proteins destined for the ER membranes themselves (Palade, 1975; Sabatini and Kreibich, 1976). In the past, much attention was paid to the association between ribosomes and the ER membranes. Less interest was taken in the process of assembly of
membrane-bound polysomes and the mechanism leading to the selection of specific classes of messenger RNA for translation on ER membranes. So far, most of the studies regarding these topics have been carried out with the aid of homogenization and subfractionation in aqueous media. Recently, a nonaqueous microdissection technique, cytoplasmic zone analysis, suitable for the analysis of the assembly of ribosomal subunits in polysomes has been developed (Edstrom and Lbnn, 1976). This technique measures the distribution of RNA in cytoplasm as related to its distance from the nuclear envelope and the time after labeled RNA precursor administration. This permits conclusions to be drawn with regard to the flow of RNA-containing particles through the cytoplasm from the nuclear envelope in intact cells in living animals. Since the dissection technique is applied to fixed cells, several of the artifacts that can arise in conventional fractionation procedures are eliminated. This technique has earlier shown gradients of newly synthesized ribosomal subunits extending through the cytoplasm. The duration and the pharmacological properties of these gradients were influenced by associations in which the ribosomal subunits engaged (Lonn and Edstrom, 1976, 1977a). The technique of cytoplasmic zone analysis is used in this paper to examine the cytoplasmic distribution of an RNA with properties of a messenger RNA coding for secretory proteins. The salivary gland cells of the midge Chironomus tentans provide suitable material for these analyses. These are secretory cells in which at least 80% of protein synthesis is accounted for by synthesis of secretory proteins (Doyle and Laufer, 1969; Pankow, Lezzi and Holderegger-Mahling, 1976). It seems probable that the synthesis of salivary secretion is largely determined by two large tissue-specific puffs, Balbiani rings 1 and 2, on chromosome 4 (Beermann, 1961; Grossbach, 1969). In agreement, the RNA synthesized in the Balbiani rings, 75s RNA in size (Daneholt, 1972), has been shown to have properties of a messenger RNA-that is, it contains poly(A) (Edstrom and Tanguay, 1974) and is located in polysomes (Daneholt, Andersson and Fagerlind, 1977). Moreover, Balbiani ring 75s RNA is associated with microsomal membranes, and the preservation of this association in vitro does not depend upon ribosomes or polysomal structures (Lonn, 1977). After a pulse of labeled RNA precursor to the living animal, newly synthesized Balbiani ring 75s RNA is found mainly in the cytoplasm located closer to the nucleus, with a gradient of decreasing abundance toward the periphery of the cell. This suggests that there is a restricted peripheral spread
Cell 728
of newly synthesized 75s RNA. The duration and pharmacological properties of this gradient may provide a unique model for investigating the association of a defined messenger RNA with the membranes of the endoplasmic reticulum in vivo. Results Newly Synthesized 75s RNA Appears Mainly in the Cytoplasm Adjoining the Nucleus The cytoplasm of fixed salivary gland cells was dissected into three concentric zones, distinguished by their distance from the nuclear envelope (for details, see Experimental Procedures). Each zone corresponds to about one third of the cytoplasmic volume. The 75s RNA exists in large amounts in the nuclear sap (Lambert et al., 1973), which is the reason why it is difficult to avoid contamination of the cytoplasm surrounding the nucleus (inner cytoplasmic zone). This cytoplasm was therefore discarded. The remaining two samples (middle and outer zones) were dissolved in detergent solution, and the RNA was separated by gel electrophoresis to quantitate the different la-
CPM
A ES
4s
50 SLICE NO.
Figure 1. Electrophoretic of Chironomus Salivary
Analysis of RNA from Gland Cells
Total
Cytoplasm
Larvae were injected with 25 &i 3H-uridine 18 hr before sacrifice. The salivary glands were fixed in ethanol/acetic acid (39 by volume). Nucleus and cytoplasm were separated by microdissection of the cells in a nonaqueous medium with the help of two glass needles attached to a de Fonbrune micromanipulator. The RNA was extracted from the total cytoplasm of 12 cells, precipitated with ethanol and analyzed in a slab bed gel of 1% agarose (for details, see Experimental procedures). Arrows indicate the position of Balbiani ring 75s RNA and 4s RNA. The shaded areas show the radioactivity due to Balbiani ring 7% RNA and 45 RNA, respectively. Moreover, the electrophoretic separation shows the two ribosomal RNA (28s RNA and 1% RNA) peaks, as well as a heterogeneous material migrating between 15-755 RNA.
beled RNA species present. This simple procedure reduces the possibilities of irregular losses. Balbiani ring 75s RNA is one of the major cytoplasmic nonribosomal RNA species. It appears as a well defined peak in gel electrophoretic separations of total cytoplasmic RNA (Figure 1; Daneholt and Hosick, 1973). The chromosomal origin of the material designated 75s RNA has earlier been firmly established with cytological hybridizations showing that this material hybridizes to the two large tissue-specific puffs, Balbiani rings 1 and 2, located on chromosome 4 (Lambert, 1973; Lambert and Edstrom, 1974). Electrophoretic separations of RNA extracted from the middle and outer cytoplasmic zones of an animal sacrificed 90 min after injection are shown in Figure 2. The middle cytoplasmic zone contains a well defined 75s RNA peak which, however, is hardly visible in the outer cytoplasmic zone. Thus the newly synthesized 75s RNA appears mainly in the cytoplasm located closer to the nucleus, with a gradient of decreasing abundance toward the periphery of the cell. In animals sacrificed 60 min after injection or earlier, it was not possible to detect labeled 7% RNA in either outer or middle cytoplasmic zones. This, however, does not rule out that some labeled 75s RNA has already appeared in the inner cytoplasmic zone discarded in the present analysis. The amounts of radioactivity in the 75s RNA and 4s RNA species were determined from the gel electrophoretic separation of the different cytoplasmic zones (hatched areas of the 75s RNA and 45 RNA peaks in Figures 1 and 2). Labeled 4s RNA serves as a volume marker, since it distributes evenly in the cytoplasm of fixed cells (Edstrom and Lbnn, 1976). The distribution of labeled 75s RNA in cytoplasm of four different animals sacrificed 90 min after RNA precursor injection is also presented in Figure 2. The staple diagrams show, in agreement with the electrophoretic separations, that the newly synthesized 75s RNA appears mainly in the cytoplasm surrounding the nucleus, with a gradient of decreasing quantity toward the periphery of the cell. The diagram also shows that there is some variation in the labeling kinetics of cytoplasmic 75s RNA between different animals. To reduce this variation, animals of similar age and size were used. Larvae were sacrificed, and salivary glands were prepared for analysis at various times between 90 min and 6 days after RNA precursor injection. Four experiments were performed at each time point, and one animal was analyzed in each experiment. Gradients in amounts of labeled 75s RNA are best expressed as values for one zone with respect to the other. Thus when the results of the four animals
Delayed
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75s
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729
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examined are tabulated together, the value of the middle zone is given an arbitrary value and the outer cytoplasmic zone normalized accordingly. In addition, in animals sacrificed 3 hr after RNA precursor injection, there is distinctly less labeled 7% RNA in the periphery of the cytoplasm as compared with the central cytoplasm (Figure 3). This gradient lasts for 2 days, although it is less pronounced at later time points. There is no detectable gradient 6 days after RNA precursor injection. Pretreatment with Cycloheximide Prevents the Formation of the 75s RNA Gradient Cycloheximide is an efficient inhibitor of protein synthesis. It stops the process of polypeptide chain elongation (Godchaux, Adamson and Herbert, 1967) and is often used to prevent ribosomal run off during polysome extraction. This drug is therefore a convenient tool for investigating whether the 7% RNA gradient is caused by a diffusion restriction of the newly synthesized 755 RNA. Such a diffusion restriction could result from an early or immediate association of 75s RNA in cytoplasmic structures involved in protein synthesis. If so, a pretreatment with cycloheximide might prevent the formation of the gradient. In Chironomus larvae treated with cycloheximide, there is extensive export of labeled 75s RNA and 4s RNA to cytoplasm, whereas ribosomal RNA export is inhibited (Lbnn and Edstrom, 1977b). Animals were kept in culture medium containing cycloheximide (IO pg/ml) for 6 hr to inhibit protein synthesis (Lonn and Edstrbm, 1977b). They were then injected with RNA precursor and kept for a further 90 min in the continued presence of the drug before sacrifice. Zone analysis revealed that in these animals, labeled 75s RNA distributes evenly in the cytoplasm-that is, there is no gra-
50 Figure 2. Electrophoretic of Cytoplasm Obtained
C-P
I L I1 LL
Analysis of RNA from by Microdissection
Concentric
Zones
Larvae were injected with 25 &i 3H-uridine 90 min before sacrifice. The fixed salivary gland cytoplasm was dissected into concentric zones. The RNA was extracted and analyzed in 1% agarose. The letters (M) and (0) stand for the middle and outer cytoplasmic zones. The inner cytoplasmic zone was not analyzed (for details, see Experimental Procedures). The shaded areas show the radioactivity due to Balbiani ring 755 RNA and 4s RNA, respectively. Moreover, the electrophoretic separations show an 18s ribosomal RNA peak, whereas labeled 28s RNA has not yet appeared (Edstrom and Tanguay, 1974). The histograms show the distribution of labeled 75s RNA in the cytoplasm of four animals. The radioactivity in 755 RNA was divided by that in 4s RNA for each zone. The 45 RNA serves as a volume marker and has been found to distribute evenly in the cytoplasm of fixed cells (Edstrom and Lonn, 1976). The cytoplasm of the inner zones which were not analyzed are marked with broken lines. The letters (C)and (P) stand for the center and the periphery of the cytoplasm, respectively.
Cell 730
75 s/4 s
90MIN
75 s/4s
3H
18 H
2 DAYS
CYCLOHEXIMIDE
6 DAYS
90MlN
3H
18 H
I
I
I! Figure 3. Summary RNA in Cytoplasmic Injection
Diagram of the Distribution of Labeled 7% Zones at Different Times after RNA-Precursor
The times give the interval between RNA precursor injection and sacrifice of the animals. The amounts of radioactive 755 RNA and 45 RNA in the different zones were determined by gel electrophoresis (see Figures 1 and 2). The radioactivity in 7% RNA was divided by that in 4s RNA. The 4s RNA serves as a volume marker and has been found to distribute evenly in the cytoplasm of fixed cells (Edstrom and Lonn, 1976) (for other details, see Experimental Procedures). In all cases, the middle zones have been given the arbitrary value of 1 and the outer zones have been normalized on a linear scale. The cytoplasm of the inner zones which were not analyzed are marked with broken lines. The letters (C) and (P) stand for the center and the periphery of the cytoplasm, respectively. In each experiment, one animal was analyzed. The columns in the diagram represent the mean of values for four animals sacrificed at the different times. The bars give the SEM of the outer cytoplasmic zones.
dient formation (Figure 4A; see Figure 3 for untreated animals). Animals were also sacrificed 3 and 18 hr after RNA precursor injection. In neither case could a radioactivity gradient in 75s RNA be detected. Thus in animals pretreated with cycloheximide, there is no restricted mobility in cytoplasm for the newly synthesized 75s RNA. Experiments were also performed with explanted salivary glands. Animals in normal culture medium were sacrificed 90 min after RNA precursor injection; one salivary gland was placed in Cannon’s incubation medium (Ringborg and Rydlander, 1971) containing cycloheximide (500 pg/ml), and the control sister gland was placed in the same medium without the drug. Separate control experiments showed that total protein synthesis was inhibited to 91% of the untreated sister gland during a 30 min incorporation after a preincubation of the gland for 15 min. (For these determinations, the salivary glands were incubated in medium containing 3H-leucine, and the hot trichloroacetic acid-precipitable activity was determined.) Zone analysis revealed that labeled 75s RNA distributes evenly in the cytoplasm (Figure 5A) in the drugtreated salivary gland. This is in striking contrast to
C-P
Figure 4. Effect Gradients
of
Protein
Synthesis
Inhibitors
in 755
RNA
(A) Larvae were pretreated with cycloheximide (IO pg/ml) added to the culture medium for 6 hr, injected with labeled RNA precursor (Lonn and Edstrom, 1977b) and cultivated in the continued presence of the drug for further 90 min, 3 hr or 18 hr before sacrifice. (B) Larvae injected with labeled RNA precursor were placed in culture medium containing cycloheximide (10 +g/ml) 24 hr later and sacrificed after further cultivation for 24 hr. The 75514s RNA label ratios in the different cytoplasmic zones were determined as described in Figure 3. In each experiment, one animal was analyzed. The columns in the diagram represent the mean of values for four animals. For symbols and other details, see Figure 3.
the sister control salivary gland in which the labeled 75s RNA is present mainly in the central cytoplasm. The results are in good agreement with the in vivo experiments. Control experiments were performed in which animals injected with labeled RNA precursor were placed in culture medium containing cycloheximide (10 pg/ml) 24 hr later and then sacrificed after further cultivation for 24 hr. In these animals, labeled 755 RNA appeared to a large degree in the central cytoplasm (Figure 4B). This distribution closely resembles that which occurs in untreated animals (see Figure 3). Further experiments were performed where animals were sacrificed 18 hr after RNA precursor injection, and the salivary glands were placed in Cannon’s incubation medium for an additional 45 min. One gland was placed in incubation medium containing cycloheximide (500 pg/ml), whereas the sister control gland was placed in medium without the drug. Zone analysis showed that the addition of cycloheximide had no effect on the cytoplasmic distribution of labeled 75s RNA as compared with the untreated sister gland (Figure 5B). Thus the treatment with
Delayed 731
Flow
a
of Balbiani
Ring 755 RNA
75 s/45
Y CONTROL CYCLOHEXIMIDE
90 + 45 MIN
CONTROL
1
CYCLOHEXIMIDE
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I I
I
I
t
&I
Ld
C-P
Y CONTROL
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CYCLOHEXIMIDE
18H+45 MIN
b/
CONTROL
CYCLOHEXIMIDE
18H
I I I I I I I I I
-
I I I I
+-I
C
L IL
Figure
6. Effect
,L L
-
I I I
I
I I
I I
I
1
of 5-Fluorouridine
1;
1
I
IL
on 75s
RNA Gradients
Larvae pretreated with 5-fluorouridine (injection of 5 fig; overall body concentration about 200 pg/ml) received a second injection with 20 PCi 3H-adenosine (Lonn, 1976) 6 hr later. The times give the interval between RNA precursor injection and sacrifice of the animals. The 75S/4S RNA label ratios in the different cytoplasmic zones were determined as described in Figure 3. In each experiment, one animal was analyzed. The columns in the diagram represent the mean of values for four animals sacrificed at the different times. For symbols and other details, see Figure 3.
“t.. Figure 5. Effect of Cycloheximide on 755 RNA Gradients
3H
I
t
q
5-FLUOROURIDINE
75S4s /
b/
in Explanted
Salivary
Glands
Larvae were injected with labeled RNA precursor (A) 90 min or(B) 18 hr before sacrifice. One salivary gland was placed in Cannon’s modified culture medium (control) (Ringborg and Rydlander, 1971), and the sister salivary gland was placed in the same medium supplemented with cycloheximide (500 pg/ml). After 45 min, both glands were fixed and prepared for analysis. The 75% 4s RNA label ratios in the different cytoplasmic zones were determined as described in the legend to Figure 2. (a) and (b) denote two different animals. The cytoplasm of the inner zones which were not analyzed are marked with broken lines. The letters (C) and (P) stand for the center and the periphery of the cell, respectively.
cycloheximide does not annihilate a preformed gradient extending through the cytoplasm. Thus pretreatment with cycloheximide prevents the normal association of newly synthesized 75s RNA with cytoplasmic structures involved in protein synthesis. 75s RNA Gradient Forms in the Absence of Ribosomal Subunit Export The effect of 5-fluorouridine in dipteran salivary glands mimics that of low doses of actinomycin D in cultured mammalian cells (Lbnn, 1978). There is
a selective inhibition of the export of ribosomal RNA to cytoplasm with little or no effect on total protein synthesis. It has been established that in drug-treated animals, the cytoplasm receives large amounts of labeled 7% RNA and 4s RNA. Animals pretreated with 5-fluorouridine were injected with labeled RNA precursor and then sacrificed after further cultivation for 90 min, 3 hr or 18 hr. Cytoplasmic zone analysis revealed that also after this treatment, labeled 75s RNA appeared mainly in the central cytoplasm with smaller amounts in the periphery of the cell (Figure 6). This mirrors the distribution in non-drug-treated animals (Figure 3). Thus there is no indication that the formation of the 75s RNA gradient depends upon simultaneous export of ribosomal subunits. Discussion The concept of gradients has been used widely in biology. Rarely, however, have gradients been detected experimentally. In investigating the presence or absence of gradients in cytoplasm, the technique of zone analysis has many advantages,
Cell 732
which are shown in this paper. Balbiani ring 7% RNA has properties of a messenger RNA probably coding for secretory proteins. This RNA appears during the first hours after RNA precursor injection, mainly in the cytoplasm located closer to the nuclear envelope with a gradient of decreasing amounts toward the periphery of the cell. This gradient lasts for at least 2 days, but not as much as 6 days. At 6 days there is an even distribution in the cytoplasm of labeled 75s RNA. The injection procedure labels RNA during 1-2 days, but most intensely during the first 18 hr (Lbnn and Edstrom, 1977a). The half-life of cytoplasmic 75s RNA is about 20 hr (Edstrom et al., 1978). Thus the 6 day injected animals should depict the distribution mainly for older 75s RNA. There is no gradient 6 days after precursor injection. This indicates that the 75s RNA synthesized during the last 2 days is located closer to the nuclear envelope than the older 75s RNA, and in turn suggests a delay in the peripheral spread of 75s RNA through cytoplasm. A pretreatment of the living animals with cycloheximide prevents the formation of the 75s RNA gradient. The 75s RNA exported to cytoplasm in these animals distributes according to cytoplasmic volume. This mirrors the distribution of 75s RNA in cytoplasm of normally cultured animals killed 6 days after RNA precursor injection. Control experiments showed that the drug does not annihilate a preformed gradient extending through the cytoplasm. Thus the effect of cycloheximide in animals pretreated with the drug is to prevent the normal association of newly synthesized 75s RNA in cytoplasmic structures involved in protein synthesis. It is very probable, therefore, that the 75s RNA gradient detected in salivary gland cytoplasm of normally cultured animals shortly after precursor injection is the result of a diffusion restriction. More than 90% of the cytoplasmic 75s RNA is associated with microsomal membranes 24 hr after RNA precursor injection (Lonn, 1977). It is probable, therefore, that the delay in the peripheral spread of the 75s RNA through the cytoplasm is caused by an association of the 75s RNA with the ER membranes. This may occur as single particles and/or as parts of polysomes. It is known that the 75s RNA-membrane interaction may be preserved in vitro independently of ribosomes or polysomal structures (Lonn, 1977). According to the experiments with cycloheximide, however, functional polysomes may be needed for the initial formation of this interaction. The association of 75s RNA to ER membranes must occur in the cytoplasm surrounding the nucleus early or immediately after nuclear release.
This is because the 75S/4S RNA label ratio is much higher in the central than in the peripheral cytoplasm. One possibility is that the association already takes place on the nuclear envelope. In yeast, it has been proposed that the poly(A)-containing RNA emerges into the cytoplasm in close relationship with the ER membranes (Shiokawa and Pogo, 1974). In the presence of 5-fluorouridine, 75s RNA is released into the cytoplasm while ribosomal RNA is not (Lonn, 1978). In addition, in animals pretreated with this drug, the newly synthesized 75s RNA forms a gradient with higher values in the central than in the peripheral cytoplasm. Thus the early or immediate association of 755 RNA to ER membranes does not depend upon the simultaneous export of ribosomal subunits. Experimental
Procedures
Animals and Labeling Conditions Late fourth instar larvae of Chironomus tentans (7-9 weeks old), weighing around 25 mg, were used. The animals were cultured as described earlier (Lonn and Edstrom, 1977a). For labeling of the RNA, the animals were injected with 25 &i of 3H-uridine (42-50 Ci/mmole) dissolved in 1 ~1 of 0.67% NaCl, 0.04% KCI. The injection was performed with a micropipette, held with a de Fonbrune micromanipulator, under the control of a stereomicroscope. The tip of the pipette is about IO ym wide. In the experiments with 5fluorouridine, the animals were given 20 j&i of 3H-adenosine (16-20 Ci/mmole) instead of uridine. Digestion with ribonuclease completely degraded the labeled cytoplasmic peaks whether uridine or adenosine was used as labeled RNA precursor (Edstrom and Tanguay, 1974; Lonn, 1978). Dissection of Salivary Gland Cytoplasm The excised salivary glands were fixed for 5 min at +4”C in freshly prepared ethanol/glacial acetic acid, 3:l by volume, followed by 2 x 15 min rinses in cold 70% ethanol, and finally stored in a mixture of glycerol/ethanol, 1:i by volume. The dissection of the cells took place in a droplet of the glycerol/ethanol mixture. The cytoplasm of the cells was dissected into concentric zones distinguished by its distance from the nuclear envelope (Edstrom and Lonn, 1976). The dissection took place in an oil chamber arrangement with the aid of two glass needles attached to a de Fonbrune micromanipulator. Flat cells of uniform size with central nuclei were selected. The outer part of the cytoplasm was removed first, and then the intermediate parts of the cytoplasm. Each part corresponds to about one third of the cytoplasmic volume. The remaining one third of the cytoplasmic volume surrounding the nucleus was discarded. The width of the zones was 20-30 pm. For each analysis, 12 cells from one pair of glands were dissected, and the material of the outer and middle cytoplasmic zones was pooled. The resulting two samples were always analyzed in parallel. Extraction and Electrophoretic Fractionation of RNA The cytoplasmic samples were extracted in 200 PI of a preincubated solution of 0.02 M Tris-HCI (pH 7.4), 0.5% sodium dodecylsulphate (SDS) and nuclease-free pronase (1 mg/ml), and with 30 pg E. coli carrier RNA. After 5 min at +25”C, the RNA was precipitated by the addition of NaCl to a final concentration of 0.1 M and 2.5 vol of cold ethanol. After storage overnight at -2o”C, the precipitate was collected by centrifugation and dissolved in 70 ~1 of 0.02 M Tris-HCI (pH 7.4) containing 0.5% SDS. The dissolved RNA was fractionated in slab bed gels of 1% agarose.
Delayed 733
Flow
of Balbiani
Ring
75s
RNA
Agarose slab bed gels were made by dissolving agarose in buffer [0.2% SDS, 0.02 M Nacl, 0.002 M EDTA in Tris-HCI (pH 7.4)] in a boiling water bath and subsequently pouring the solution into a slab mold. The gel was ready for use after 45 min at +4”C. The two samples of one experiment were applied in 15 x 2 x 2 mm throughs, respectively. The separation time was about 2.5 hr at 40 V/12 cm. At the end of the run, the gel slab was treated with cold 5% trichloroacetic acid for 2 x 50 min and finally rinsed in water before slicing. The slices (1 .I mm wide) were each placed in disposable vials containing 10 ml scintillation cocktail with 3% Soluene-100 (Edstrdm and Lonn, 1976) and placed at +37”C overnight, after which they were ready for counting in a Packard scintillation counter.
Lambert, B. (1973). Tracing of RNA from a puff in the polytene chromosomes to the cytoplasm in Chironomus tentans salivary gland cells. Nature 242, 51-53.
Materials Commercial sources of chemicals were as follows: The Radiochemical Centre (Amersham, England) for 5,6-3H-uridine (42-50 Ci/mmole) and 5-3H-adenosine (18-20 Cilmmole); Calbiochem (San Diego, California) for 5-fluorouridine and pronase (nucleasefree); Bausch and Lomb (Consumer Products Division, Rochester, New York) for agarose; Serva (Heidelberg, Germany) for sodium dodecylsulphate; Sigma Chemicals (St. Louis, Missouri) for cycloheximide.
Lonn, U. (1978). Differential inhibitory effect of 5-fluorouridine on RNA labelling in dipteran salivary gland cells. Biochim. Biophys. Acta, in press.
Acknowledgments The author is indebted to Kerstin Spetz for skillful technical assistance, to Chana Szpiro for culturing the animals and to Hannele Jansson for typing the manuscript. The present work was supported by grants from the Swedish Cancer Society and Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
November
16,1977;
revised
January
19,1978
References Beermann, W. (1961). Ein Balbiani-Ring als cheldrusenmutation. Chromosoma 72, l-25. Daneholt, B. (1972). Giant New Biol. 240, 229-232.
RNA transcript
Daneholt, B. and Hosick, H. (1973). RNA from a discrete chromosome cytoplasm in Chironomus tentans. 442-446.
Locus
in a Balbiani
einer ring.
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Evidence for transport of 75 S region via nuclear sap to Proc. Nat. Acad. Sci. USA 70,
Daneholt, B., Andersson, K. and Fagerlind, M. (1977). Large-sized polysomes in Chironomus tentans salivary glands and their relation to Balbiani ring 75 S RNA. J. Cell Biol. 73, 149-160. Doyle, D. and Laufer, H. (1969). Sources of larval salivary gland secretion in the dipteran Chironomus tentans. J. Cell Biol. 40, 6179. Edstrom, J.-E. and Tanguay, R. (1974). Cytoplasmic ribonucleic acids with messenger characteristics in salivary glands of Chironomus fentans. J. Mol. Biol. 84, 569-583. Edstrom, J.-E. and Lonn, U. (1976). RNA flow studied by micromanipulation.
Cytoplasmic zone analysis. J. Cell Biol. 70, 562-572.
Edstrdm, J.-E., Lindgren, S., Lonn, U. and Rydlander, L. (1978). Balbiani ring RNA and half-life in nucleus and cytoplasm of Chironomus tentans salivary gland cells. Chromosoma, in press. Godchaux, W., Ill, Adamson, S. D. and Herbert, E. (1967). Effect of cycloheximide on polyribosome function in reticulocytes. J. Mol. Biol. 27, 57-72. Grossbach, U. (1969). Chromosomen-Aktivitat Zell-differenzierung in den Speicheldrusen mus. ChromosomaPB, 136-187.
von
und biochemische Camptochirono-
Lambert, B., Edstrbm, quences in cytoplasmic gland cells. Mol. Biol.
J.-E. (1974). Balbiani ring nucleotide se75 S RNA of Chironomus tentans salivary Reports 1, 457-464.
Lambert, B., Daneholt, 8.. Edstrbm, J.-E., Egyhazi, E. and Ringborg, U. (1973). Comparison between chromosomal and nuclear sap RNA from Chironomus tentans salivary gland cells by RNA/ DNA hybridization. Exp. Cell Res. 76, 381-389. Lonn, U. (1977). the membranes 631.
Direct association of the endoplasmic
Lonn, U. and Edstrbm, J.-E. the heavy ribosomal subunit 573-580.
of Balbiani reticulum.
(1976). Mobility in a secretory
ring 75 S RNA with Nature 270, 630-
restriction in viva of cell. J. Cell Biol. 70,
Lonn, U. and Edstrom, J.-E. (1977a). Movementsand associations of ribosomal subunits in a secretory cell during growth inhibition by starvation. J. Cell Biol. 73, 696-704. Lonn, U. and Edstrom, J.-E. (1977b). Protein and export of ribosomal subunits. Biochim. 677-679.
synthesis Biophys.
Palade, protein
of the
G. E. (1975). Intracellular aspects synthesis. Science 189, 347-358.
inhibitors Acta 475, process
of
Pankow, W., Lezzi, M. and Holderegger-Mahling, I. (1976). Correlated changes of Balbiani ring expansion and secretory protein synthesis in larval salivary glands of Chironomus fentans. Chromosoma 58, 137-I 53. Ringborg, U. and Rydlander, L. (1971). Nucleolar derived ribonucleic acid in chromosomes, nuclear sap and cytoplasm of Chironomus tentans salivary gland cells. J. Cell Biol. 57, 355-368. Sabatini, D. D. and Kreibich, G. (1976). Functional specialization of membrane-bound ribosomes in eukaryotic cells. In Enzymes of Biological Membranes, 2, A. Martonosi, ed. (New York: Plenum), pp. 531-579. Shiokawa, K. and Pogo, A. 0. (1974). The role of cytoplasmic membranes in controlling the transport of nuclear messenger RNA and initiation of protein synthesis. Proc. Nat. Acad. Sci. USA 71, 2658-2662.
April
1978,
Copyright
0 1978 by MIT
Delayed Flow-Through Cytoplasm of Newly Synthesized Balbiani Ring 7% RNA U. Lonn Department of Histology Karolinska Institute S-104 01 Stockholm 60, Sweden
Summary With a nonaqueous microdissection technique, the cytoplasm of Chironomus salivary gland cells can be separated into concentric zones situated at increasing distances from the nuclear envelope. This dissection technique is used here to investigate the cytoplasmic distribution of 75s RNA of Balbiani ring origin. The Balbiani ring 7% RNA has properties of a messenger RNA coding for secretory proteins. After a pulse of RNA precursor to the living animal, labeled Balbiani ring 75s RNA is found mainly in the cytoplasm located closer to the nuclear envelope, with smaller amounts toward the periphery of the cell. This gradient, initially very steep, lasts for at least 2 days, but less than 6 days. Experiments with 5fluorouridine indicate that the formation of the gradient does not depend upon simultaneous export of ribosomal subunits. After a pretreatment of the animals with the protein synthesis inhibitor cycloheximide, however, newly synthesized 7% RNA distributes evenly in the cytoplasm-that is, this treatment prevents the formation of the 75s RNA gradient. The gradient in salivary glands of normally cultured animals is therefore likely to be the result of diffusion restriction of the labeled 75s RNA. Thus the 75s RNA located closer to the nuclear envelope is the most recently exported 755 RNA. An explanation of these results is that the 75s RNA associates with the membranes of the endoplasmic reticulum early or immediately after nuclear release. This association should occur in the cytoplasm surrounding the nucleus and may occur either as single particles and/or as parts of polysomes. Introduction Proteins destined for export from the cell, such as secretory proteins, are synthesized on polysomes associated with the membranes of the endoplasmic reticulum (ER). Such polysomes also synthesize proteins that are destined for inclusion in other membrane-bound organelles as well as proteins destined for the ER membranes themselves (Palade, 1975; Sabatini and Kreibich, 1976). In the past, much attention was paid to the association between ribosomes and the ER membranes. Less interest was taken in the process of assembly of
membrane-bound polysomes and the mechanism leading to the selection of specific classes of messenger RNA for translation on ER membranes. So far, most of the studies regarding these topics have been carried out with the aid of homogenization and subfractionation in aqueous media. Recently, a nonaqueous microdissection technique, cytoplasmic zone analysis, suitable for the analysis of the assembly of ribosomal subunits in polysomes has been developed (Edstrom and Lbnn, 1976). This technique measures the distribution of RNA in cytoplasm as related to its distance from the nuclear envelope and the time after labeled RNA precursor administration. This permits conclusions to be drawn with regard to the flow of RNA-containing particles through the cytoplasm from the nuclear envelope in intact cells in living animals. Since the dissection technique is applied to fixed cells, several of the artifacts that can arise in conventional fractionation procedures are eliminated. This technique has earlier shown gradients of newly synthesized ribosomal subunits extending through the cytoplasm. The duration and the pharmacological properties of these gradients were influenced by associations in which the ribosomal subunits engaged (Lonn and Edstrom, 1976, 1977a). The technique of cytoplasmic zone analysis is used in this paper to examine the cytoplasmic distribution of an RNA with properties of a messenger RNA coding for secretory proteins. The salivary gland cells of the midge Chironomus tentans provide suitable material for these analyses. These are secretory cells in which at least 80% of protein synthesis is accounted for by synthesis of secretory proteins (Doyle and Laufer, 1969; Pankow, Lezzi and Holderegger-Mahling, 1976). It seems probable that the synthesis of salivary secretion is largely determined by two large tissue-specific puffs, Balbiani rings 1 and 2, on chromosome 4 (Beermann, 1961; Grossbach, 1969). In agreement, the RNA synthesized in the Balbiani rings, 75s RNA in size (Daneholt, 1972), has been shown to have properties of a messenger RNA-that is, it contains poly(A) (Edstrom and Tanguay, 1974) and is located in polysomes (Daneholt, Andersson and Fagerlind, 1977). Moreover, Balbiani ring 75s RNA is associated with microsomal membranes, and the preservation of this association in vitro does not depend upon ribosomes or polysomal structures (Lonn, 1977). After a pulse of labeled RNA precursor to the living animal, newly synthesized Balbiani ring 75s RNA is found mainly in the cytoplasm located closer to the nucleus, with a gradient of decreasing abundance toward the periphery of the cell. This suggests that there is a restricted peripheral spread
Cell 728
of newly synthesized 75s RNA. The duration and pharmacological properties of this gradient may provide a unique model for investigating the association of a defined messenger RNA with the membranes of the endoplasmic reticulum in vivo. Results Newly Synthesized 75s RNA Appears Mainly in the Cytoplasm Adjoining the Nucleus The cytoplasm of fixed salivary gland cells was dissected into three concentric zones, distinguished by their distance from the nuclear envelope (for details, see Experimental Procedures). Each zone corresponds to about one third of the cytoplasmic volume. The 75s RNA exists in large amounts in the nuclear sap (Lambert et al., 1973), which is the reason why it is difficult to avoid contamination of the cytoplasm surrounding the nucleus (inner cytoplasmic zone). This cytoplasm was therefore discarded. The remaining two samples (middle and outer zones) were dissolved in detergent solution, and the RNA was separated by gel electrophoresis to quantitate the different la-
CPM
A ES
4s
50 SLICE NO.
Figure 1. Electrophoretic of Chironomus Salivary
Analysis of RNA from Gland Cells
Total
Cytoplasm
Larvae were injected with 25 &i 3H-uridine 18 hr before sacrifice. The salivary glands were fixed in ethanol/acetic acid (39 by volume). Nucleus and cytoplasm were separated by microdissection of the cells in a nonaqueous medium with the help of two glass needles attached to a de Fonbrune micromanipulator. The RNA was extracted from the total cytoplasm of 12 cells, precipitated with ethanol and analyzed in a slab bed gel of 1% agarose (for details, see Experimental procedures). Arrows indicate the position of Balbiani ring 75s RNA and 4s RNA. The shaded areas show the radioactivity due to Balbiani ring 7% RNA and 45 RNA, respectively. Moreover, the electrophoretic separation shows the two ribosomal RNA (28s RNA and 1% RNA) peaks, as well as a heterogeneous material migrating between 15-755 RNA.
beled RNA species present. This simple procedure reduces the possibilities of irregular losses. Balbiani ring 75s RNA is one of the major cytoplasmic nonribosomal RNA species. It appears as a well defined peak in gel electrophoretic separations of total cytoplasmic RNA (Figure 1; Daneholt and Hosick, 1973). The chromosomal origin of the material designated 75s RNA has earlier been firmly established with cytological hybridizations showing that this material hybridizes to the two large tissue-specific puffs, Balbiani rings 1 and 2, located on chromosome 4 (Lambert, 1973; Lambert and Edstrom, 1974). Electrophoretic separations of RNA extracted from the middle and outer cytoplasmic zones of an animal sacrificed 90 min after injection are shown in Figure 2. The middle cytoplasmic zone contains a well defined 75s RNA peak which, however, is hardly visible in the outer cytoplasmic zone. Thus the newly synthesized 75s RNA appears mainly in the cytoplasm located closer to the nucleus, with a gradient of decreasing abundance toward the periphery of the cell. In animals sacrificed 60 min after injection or earlier, it was not possible to detect labeled 7% RNA in either outer or middle cytoplasmic zones. This, however, does not rule out that some labeled 75s RNA has already appeared in the inner cytoplasmic zone discarded in the present analysis. The amounts of radioactivity in the 75s RNA and 4s RNA species were determined from the gel electrophoretic separation of the different cytoplasmic zones (hatched areas of the 75s RNA and 45 RNA peaks in Figures 1 and 2). Labeled 4s RNA serves as a volume marker, since it distributes evenly in the cytoplasm of fixed cells (Edstrom and Lbnn, 1976). The distribution of labeled 75s RNA in cytoplasm of four different animals sacrificed 90 min after RNA precursor injection is also presented in Figure 2. The staple diagrams show, in agreement with the electrophoretic separations, that the newly synthesized 75s RNA appears mainly in the cytoplasm surrounding the nucleus, with a gradient of decreasing quantity toward the periphery of the cell. The diagram also shows that there is some variation in the labeling kinetics of cytoplasmic 75s RNA between different animals. To reduce this variation, animals of similar age and size were used. Larvae were sacrificed, and salivary glands were prepared for analysis at various times between 90 min and 6 days after RNA precursor injection. Four experiments were performed at each time point, and one animal was analyzed in each experiment. Gradients in amounts of labeled 75s RNA are best expressed as values for one zone with respect to the other. Thus when the results of the four animals
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examined are tabulated together, the value of the middle zone is given an arbitrary value and the outer cytoplasmic zone normalized accordingly. In addition, in animals sacrificed 3 hr after RNA precursor injection, there is distinctly less labeled 7% RNA in the periphery of the cytoplasm as compared with the central cytoplasm (Figure 3). This gradient lasts for 2 days, although it is less pronounced at later time points. There is no detectable gradient 6 days after RNA precursor injection. Pretreatment with Cycloheximide Prevents the Formation of the 75s RNA Gradient Cycloheximide is an efficient inhibitor of protein synthesis. It stops the process of polypeptide chain elongation (Godchaux, Adamson and Herbert, 1967) and is often used to prevent ribosomal run off during polysome extraction. This drug is therefore a convenient tool for investigating whether the 7% RNA gradient is caused by a diffusion restriction of the newly synthesized 755 RNA. Such a diffusion restriction could result from an early or immediate association of 75s RNA in cytoplasmic structures involved in protein synthesis. If so, a pretreatment with cycloheximide might prevent the formation of the gradient. In Chironomus larvae treated with cycloheximide, there is extensive export of labeled 75s RNA and 4s RNA to cytoplasm, whereas ribosomal RNA export is inhibited (Lbnn and Edstrom, 1977b). Animals were kept in culture medium containing cycloheximide (IO pg/ml) for 6 hr to inhibit protein synthesis (Lonn and Edstrbm, 1977b). They were then injected with RNA precursor and kept for a further 90 min in the continued presence of the drug before sacrifice. Zone analysis revealed that in these animals, labeled 75s RNA distributes evenly in the cytoplasm-that is, there is no gra-
50 Figure 2. Electrophoretic of Cytoplasm Obtained
C-P
I L I1 LL
Analysis of RNA from by Microdissection
Concentric
Zones
Larvae were injected with 25 &i 3H-uridine 90 min before sacrifice. The fixed salivary gland cytoplasm was dissected into concentric zones. The RNA was extracted and analyzed in 1% agarose. The letters (M) and (0) stand for the middle and outer cytoplasmic zones. The inner cytoplasmic zone was not analyzed (for details, see Experimental Procedures). The shaded areas show the radioactivity due to Balbiani ring 755 RNA and 4s RNA, respectively. Moreover, the electrophoretic separations show an 18s ribosomal RNA peak, whereas labeled 28s RNA has not yet appeared (Edstrom and Tanguay, 1974). The histograms show the distribution of labeled 75s RNA in the cytoplasm of four animals. The radioactivity in 755 RNA was divided by that in 4s RNA for each zone. The 45 RNA serves as a volume marker and has been found to distribute evenly in the cytoplasm of fixed cells (Edstrom and Lonn, 1976). The cytoplasm of the inner zones which were not analyzed are marked with broken lines. The letters (C)and (P) stand for the center and the periphery of the cytoplasm, respectively.
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I! Figure 3. Summary RNA in Cytoplasmic Injection
Diagram of the Distribution of Labeled 7% Zones at Different Times after RNA-Precursor
The times give the interval between RNA precursor injection and sacrifice of the animals. The amounts of radioactive 755 RNA and 45 RNA in the different zones were determined by gel electrophoresis (see Figures 1 and 2). The radioactivity in 7% RNA was divided by that in 4s RNA. The 4s RNA serves as a volume marker and has been found to distribute evenly in the cytoplasm of fixed cells (Edstrom and Lonn, 1976) (for other details, see Experimental Procedures). In all cases, the middle zones have been given the arbitrary value of 1 and the outer zones have been normalized on a linear scale. The cytoplasm of the inner zones which were not analyzed are marked with broken lines. The letters (C) and (P) stand for the center and the periphery of the cytoplasm, respectively. In each experiment, one animal was analyzed. The columns in the diagram represent the mean of values for four animals sacrificed at the different times. The bars give the SEM of the outer cytoplasmic zones.
dient formation (Figure 4A; see Figure 3 for untreated animals). Animals were also sacrificed 3 and 18 hr after RNA precursor injection. In neither case could a radioactivity gradient in 75s RNA be detected. Thus in animals pretreated with cycloheximide, there is no restricted mobility in cytoplasm for the newly synthesized 75s RNA. Experiments were also performed with explanted salivary glands. Animals in normal culture medium were sacrificed 90 min after RNA precursor injection; one salivary gland was placed in Cannon’s incubation medium (Ringborg and Rydlander, 1971) containing cycloheximide (500 pg/ml), and the control sister gland was placed in the same medium without the drug. Separate control experiments showed that total protein synthesis was inhibited to 91% of the untreated sister gland during a 30 min incorporation after a preincubation of the gland for 15 min. (For these determinations, the salivary glands were incubated in medium containing 3H-leucine, and the hot trichloroacetic acid-precipitable activity was determined.) Zone analysis revealed that labeled 75s RNA distributes evenly in the cytoplasm (Figure 5A) in the drugtreated salivary gland. This is in striking contrast to
C-P
Figure 4. Effect Gradients
of
Protein
Synthesis
Inhibitors
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RNA
(A) Larvae were pretreated with cycloheximide (IO pg/ml) added to the culture medium for 6 hr, injected with labeled RNA precursor (Lonn and Edstrom, 1977b) and cultivated in the continued presence of the drug for further 90 min, 3 hr or 18 hr before sacrifice. (B) Larvae injected with labeled RNA precursor were placed in culture medium containing cycloheximide (10 +g/ml) 24 hr later and sacrificed after further cultivation for 24 hr. The 75514s RNA label ratios in the different cytoplasmic zones were determined as described in Figure 3. In each experiment, one animal was analyzed. The columns in the diagram represent the mean of values for four animals. For symbols and other details, see Figure 3.
the sister control salivary gland in which the labeled 75s RNA is present mainly in the central cytoplasm. The results are in good agreement with the in vivo experiments. Control experiments were performed in which animals injected with labeled RNA precursor were placed in culture medium containing cycloheximide (10 pg/ml) 24 hr later and then sacrificed after further cultivation for 24 hr. In these animals, labeled 755 RNA appeared to a large degree in the central cytoplasm (Figure 4B). This distribution closely resembles that which occurs in untreated animals (see Figure 3). Further experiments were performed where animals were sacrificed 18 hr after RNA precursor injection, and the salivary glands were placed in Cannon’s incubation medium for an additional 45 min. One gland was placed in incubation medium containing cycloheximide (500 pg/ml), whereas the sister control gland was placed in medium without the drug. Zone analysis showed that the addition of cycloheximide had no effect on the cytoplasmic distribution of labeled 75s RNA as compared with the untreated sister gland (Figure 5B). Thus the treatment with
Delayed 731
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,L L
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1;
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RNA Gradients
Larvae pretreated with 5-fluorouridine (injection of 5 fig; overall body concentration about 200 pg/ml) received a second injection with 20 PCi 3H-adenosine (Lonn, 1976) 6 hr later. The times give the interval between RNA precursor injection and sacrifice of the animals. The 75S/4S RNA label ratios in the different cytoplasmic zones were determined as described in Figure 3. In each experiment, one animal was analyzed. The columns in the diagram represent the mean of values for four animals sacrificed at the different times. For symbols and other details, see Figure 3.
“t.. Figure 5. Effect of Cycloheximide on 755 RNA Gradients
3H
I
t
q
5-FLUOROURIDINE
75S4s /
b/
in Explanted
Salivary
Glands
Larvae were injected with labeled RNA precursor (A) 90 min or(B) 18 hr before sacrifice. One salivary gland was placed in Cannon’s modified culture medium (control) (Ringborg and Rydlander, 1971), and the sister salivary gland was placed in the same medium supplemented with cycloheximide (500 pg/ml). After 45 min, both glands were fixed and prepared for analysis. The 75% 4s RNA label ratios in the different cytoplasmic zones were determined as described in the legend to Figure 2. (a) and (b) denote two different animals. The cytoplasm of the inner zones which were not analyzed are marked with broken lines. The letters (C) and (P) stand for the center and the periphery of the cell, respectively.
cycloheximide does not annihilate a preformed gradient extending through the cytoplasm. Thus pretreatment with cycloheximide prevents the normal association of newly synthesized 75s RNA with cytoplasmic structures involved in protein synthesis. 75s RNA Gradient Forms in the Absence of Ribosomal Subunit Export The effect of 5-fluorouridine in dipteran salivary glands mimics that of low doses of actinomycin D in cultured mammalian cells (Lbnn, 1978). There is
a selective inhibition of the export of ribosomal RNA to cytoplasm with little or no effect on total protein synthesis. It has been established that in drug-treated animals, the cytoplasm receives large amounts of labeled 7% RNA and 4s RNA. Animals pretreated with 5-fluorouridine were injected with labeled RNA precursor and then sacrificed after further cultivation for 90 min, 3 hr or 18 hr. Cytoplasmic zone analysis revealed that also after this treatment, labeled 75s RNA appeared mainly in the central cytoplasm with smaller amounts in the periphery of the cell (Figure 6). This mirrors the distribution in non-drug-treated animals (Figure 3). Thus there is no indication that the formation of the 75s RNA gradient depends upon simultaneous export of ribosomal subunits. Discussion The concept of gradients has been used widely in biology. Rarely, however, have gradients been detected experimentally. In investigating the presence or absence of gradients in cytoplasm, the technique of zone analysis has many advantages,
Cell 732
which are shown in this paper. Balbiani ring 7% RNA has properties of a messenger RNA probably coding for secretory proteins. This RNA appears during the first hours after RNA precursor injection, mainly in the cytoplasm located closer to the nuclear envelope with a gradient of decreasing amounts toward the periphery of the cell. This gradient lasts for at least 2 days, but not as much as 6 days. At 6 days there is an even distribution in the cytoplasm of labeled 75s RNA. The injection procedure labels RNA during 1-2 days, but most intensely during the first 18 hr (Lbnn and Edstrom, 1977a). The half-life of cytoplasmic 75s RNA is about 20 hr (Edstrom et al., 1978). Thus the 6 day injected animals should depict the distribution mainly for older 75s RNA. There is no gradient 6 days after precursor injection. This indicates that the 75s RNA synthesized during the last 2 days is located closer to the nuclear envelope than the older 75s RNA, and in turn suggests a delay in the peripheral spread of 75s RNA through cytoplasm. A pretreatment of the living animals with cycloheximide prevents the formation of the 75s RNA gradient. The 75s RNA exported to cytoplasm in these animals distributes according to cytoplasmic volume. This mirrors the distribution of 75s RNA in cytoplasm of normally cultured animals killed 6 days after RNA precursor injection. Control experiments showed that the drug does not annihilate a preformed gradient extending through the cytoplasm. Thus the effect of cycloheximide in animals pretreated with the drug is to prevent the normal association of newly synthesized 75s RNA in cytoplasmic structures involved in protein synthesis. It is very probable, therefore, that the 75s RNA gradient detected in salivary gland cytoplasm of normally cultured animals shortly after precursor injection is the result of a diffusion restriction. More than 90% of the cytoplasmic 75s RNA is associated with microsomal membranes 24 hr after RNA precursor injection (Lonn, 1977). It is probable, therefore, that the delay in the peripheral spread of the 75s RNA through the cytoplasm is caused by an association of the 75s RNA with the ER membranes. This may occur as single particles and/or as parts of polysomes. It is known that the 75s RNA-membrane interaction may be preserved in vitro independently of ribosomes or polysomal structures (Lonn, 1977). According to the experiments with cycloheximide, however, functional polysomes may be needed for the initial formation of this interaction. The association of 75s RNA to ER membranes must occur in the cytoplasm surrounding the nucleus early or immediately after nuclear release.
This is because the 75S/4S RNA label ratio is much higher in the central than in the peripheral cytoplasm. One possibility is that the association already takes place on the nuclear envelope. In yeast, it has been proposed that the poly(A)-containing RNA emerges into the cytoplasm in close relationship with the ER membranes (Shiokawa and Pogo, 1974). In the presence of 5-fluorouridine, 75s RNA is released into the cytoplasm while ribosomal RNA is not (Lonn, 1978). In addition, in animals pretreated with this drug, the newly synthesized 75s RNA forms a gradient with higher values in the central than in the peripheral cytoplasm. Thus the early or immediate association of 755 RNA to ER membranes does not depend upon the simultaneous export of ribosomal subunits. Experimental
Procedures
Animals and Labeling Conditions Late fourth instar larvae of Chironomus tentans (7-9 weeks old), weighing around 25 mg, were used. The animals were cultured as described earlier (Lonn and Edstrom, 1977a). For labeling of the RNA, the animals were injected with 25 &i of 3H-uridine (42-50 Ci/mmole) dissolved in 1 ~1 of 0.67% NaCl, 0.04% KCI. The injection was performed with a micropipette, held with a de Fonbrune micromanipulator, under the control of a stereomicroscope. The tip of the pipette is about IO ym wide. In the experiments with 5fluorouridine, the animals were given 20 j&i of 3H-adenosine (16-20 Ci/mmole) instead of uridine. Digestion with ribonuclease completely degraded the labeled cytoplasmic peaks whether uridine or adenosine was used as labeled RNA precursor (Edstrom and Tanguay, 1974; Lonn, 1978). Dissection of Salivary Gland Cytoplasm The excised salivary glands were fixed for 5 min at +4”C in freshly prepared ethanol/glacial acetic acid, 3:l by volume, followed by 2 x 15 min rinses in cold 70% ethanol, and finally stored in a mixture of glycerol/ethanol, 1:i by volume. The dissection of the cells took place in a droplet of the glycerol/ethanol mixture. The cytoplasm of the cells was dissected into concentric zones distinguished by its distance from the nuclear envelope (Edstrom and Lonn, 1976). The dissection took place in an oil chamber arrangement with the aid of two glass needles attached to a de Fonbrune micromanipulator. Flat cells of uniform size with central nuclei were selected. The outer part of the cytoplasm was removed first, and then the intermediate parts of the cytoplasm. Each part corresponds to about one third of the cytoplasmic volume. The remaining one third of the cytoplasmic volume surrounding the nucleus was discarded. The width of the zones was 20-30 pm. For each analysis, 12 cells from one pair of glands were dissected, and the material of the outer and middle cytoplasmic zones was pooled. The resulting two samples were always analyzed in parallel. Extraction and Electrophoretic Fractionation of RNA The cytoplasmic samples were extracted in 200 PI of a preincubated solution of 0.02 M Tris-HCI (pH 7.4), 0.5% sodium dodecylsulphate (SDS) and nuclease-free pronase (1 mg/ml), and with 30 pg E. coli carrier RNA. After 5 min at +25”C, the RNA was precipitated by the addition of NaCl to a final concentration of 0.1 M and 2.5 vol of cold ethanol. After storage overnight at -2o”C, the precipitate was collected by centrifugation and dissolved in 70 ~1 of 0.02 M Tris-HCI (pH 7.4) containing 0.5% SDS. The dissolved RNA was fractionated in slab bed gels of 1% agarose.
Delayed 733
Flow
of Balbiani
Ring
75s
RNA
Agarose slab bed gels were made by dissolving agarose in buffer [0.2% SDS, 0.02 M Nacl, 0.002 M EDTA in Tris-HCI (pH 7.4)] in a boiling water bath and subsequently pouring the solution into a slab mold. The gel was ready for use after 45 min at +4”C. The two samples of one experiment were applied in 15 x 2 x 2 mm throughs, respectively. The separation time was about 2.5 hr at 40 V/12 cm. At the end of the run, the gel slab was treated with cold 5% trichloroacetic acid for 2 x 50 min and finally rinsed in water before slicing. The slices (1 .I mm wide) were each placed in disposable vials containing 10 ml scintillation cocktail with 3% Soluene-100 (Edstrdm and Lonn, 1976) and placed at +37”C overnight, after which they were ready for counting in a Packard scintillation counter.
Lambert, B. (1973). Tracing of RNA from a puff in the polytene chromosomes to the cytoplasm in Chironomus tentans salivary gland cells. Nature 242, 51-53.
Materials Commercial sources of chemicals were as follows: The Radiochemical Centre (Amersham, England) for 5,6-3H-uridine (42-50 Ci/mmole) and 5-3H-adenosine (18-20 Cilmmole); Calbiochem (San Diego, California) for 5-fluorouridine and pronase (nucleasefree); Bausch and Lomb (Consumer Products Division, Rochester, New York) for agarose; Serva (Heidelberg, Germany) for sodium dodecylsulphate; Sigma Chemicals (St. Louis, Missouri) for cycloheximide.
Lonn, U. (1978). Differential inhibitory effect of 5-fluorouridine on RNA labelling in dipteran salivary gland cells. Biochim. Biophys. Acta, in press.
Acknowledgments The author is indebted to Kerstin Spetz for skillful technical assistance, to Chana Szpiro for culturing the animals and to Hannele Jansson for typing the manuscript. The present work was supported by grants from the Swedish Cancer Society and Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
November
16,1977;
revised
January
19,1978
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Edstrdm, J.-E., Lindgren, S., Lonn, U. and Rydlander, L. (1978). Balbiani ring RNA and half-life in nucleus and cytoplasm of Chironomus tentans salivary gland cells. Chromosoma, in press. Godchaux, W., Ill, Adamson, S. D. and Herbert, E. (1967). Effect of cycloheximide on polyribosome function in reticulocytes. J. Mol. Biol. 27, 57-72. Grossbach, U. (1969). Chromosomen-Aktivitat Zell-differenzierung in den Speicheldrusen mus. ChromosomaPB, 136-187.
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Lambert, B., Daneholt, 8.. Edstrbm, J.-E., Egyhazi, E. and Ringborg, U. (1973). Comparison between chromosomal and nuclear sap RNA from Chironomus tentans salivary gland cells by RNA/ DNA hybridization. Exp. Cell Res. 76, 381-389. Lonn, U. (1977). the membranes 631.
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inhibitors Acta 475, process
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Pankow, W., Lezzi, M. and Holderegger-Mahling, I. (1976). Correlated changes of Balbiani ring expansion and secretory protein synthesis in larval salivary glands of Chironomus fentans. Chromosoma 58, 137-I 53. Ringborg, U. and Rydlander, L. (1971). Nucleolar derived ribonucleic acid in chromosomes, nuclear sap and cytoplasm of Chironomus tentans salivary gland cells. J. Cell Biol. 57, 355-368. Sabatini, D. D. and Kreibich, G. (1976). Functional specialization of membrane-bound ribosomes in eukaryotic cells. In Enzymes of Biological Membranes, 2, A. Martonosi, ed. (New York: Plenum), pp. 531-579. Shiokawa, K. and Pogo, A. 0. (1974). The role of cytoplasmic membranes in controlling the transport of nuclear messenger RNA and initiation of protein synthesis. Proc. Nat. Acad. Sci. USA 71, 2658-2662.